'Plasmacytoid' dendritic cells (pDC) can regulate human T cell responses in vitro, including induction of T regulatory (T reg) cells. This suggests a role for pDC in immunological tolerance. Assessment of the functional significance of these cells in vivo has been greatly enhanced by the recent identification of the murine counterparts of human pDC. We have purified (immature) pDC from mouse lymphoid tissue (spleen) and found that their infusion (x1) into fully-allogeneic, non-immunosuppressed recipients results in marked donor-specific prolongation of vascularized organ graft survival. The effect achieved is significantly greater than that attained under identical conditions with either freshly-isolated (immature) classic myeloid (M) DC (CD8alpha-) or CD8alpha+ DC (a distinct murine DC subset, with no known human counterpart). The mechanism(s) underlying this tolerogenic effect is unknown. Based on improved understanding of how pDC regulate alloreactive T cell responses, and our own and other's previous experience of potentiating the tolerogenic effect of donor hematopoietic cells (DC) in transplant models, we postulate that long-term allograft survival and donor-specific tolerance can be achieved using this novel DC subset with inherent regulatory potential. We can obtain highly-purified pDC from mice given the DC-mobilizing agent fms-like tyrosine kinase 3 ligand (Flt3L) and can also propagate and sort pDC in adequate numbers for in vivo study. Our preliminary data show that immature pDC express novel B7 family coregulatory molecules,- programmed death ligand-1 (PD-L1) and inducible costimulator ligand (ICOSL), that may play important roles in determining the outcome of their interaction with allogeneic T cells. Significantly, freshly-isolated pDC, but not MDC, also express indoleamine 2, 3 dioxygenase (IDO), an enzyme implicated in the regulation of allogeneic T cell proliferation. Taken together, these findings suggest that pDC may be uniquely well-equipped to promote long-term graft survival. We hypothesize that signaling via novel coregulatory pathways and expression of IDO accounts for the 'superior' tolerogenicity of pDC compared with MDC and underpins the ability of these cells to subvert alloreactive T cell responses and promote transplant tolerance. While we postulate that pDC may induce T reg cells capable of inhibiting alloAg-specific effector T cell responses, we will not overlook the possibility that other, non-exclusive and perhaps temporally-dissociated mechanisms may be involved. In Aim I, we will use specific inhibitors and cells from gene knockout mice to define the molecular pathways that we hypothesize are involved in the regulation of allogeneic T cell responses by pDC compared with MDC. In Aim II, we will optimize and analyze conditions for promotion of transplant tolerance by pDC, and ascertain whether deficiency or blocking of the signaling pathways that we hypothesize may be critical for the tolerogenic effect of pDC will abrogate tolerance. In Aim III, we will determine the mechanisms that underlie the regulation of anti-donor T cell responses by pDC in graft recipients; in particular the role of T reg cells, and establishes whether the tolerogenic mechanisms induced by pDC depend on specific coregulatory pathways. The results will provide important new insight into the molecular signaling pathways that determine the comparative tolerogenic efficacy of pDC versus classic MDC and serve to guide strategies for testing of pDC in large animal and clinical organ transplantation.